Plants and seeds of hybrid corn variety CH540328

ABSTRACT

According to the invention, there is provided seed and plants of the hybrid corn variety designated CH540328. The invention thus relates to the plants, seeds and tissue cultures of the variety CH540328, and to methods for producing a corn plant produced by crossing a corn plant of variety CH540328 with itself or with another corn plant, such as a plant of another variety. The invention further relates to genetic complements of plants of variety CH540328.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appl. Ser. No.61/480,355, filed Apr. 28, 2011, the entire disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of corn breeding.In particular, the invention relates to corn seed and plants of thehybrid variety designated CH540328, and derivatives and tissue culturesthereof.

2. Description of Related Art

The goal of field crop breeding is to combine various desirable traitsin a single variety/hybrid. Such desirable traits include greater yield,better stalks, better roots, resistance to insecticides, herbicides,pests, and disease, tolerance to heat and drought, reduced time to cropmaturity, better agronomic quality, higher nutritional value, anduniformity in germination times, stand establishment, growth rate,maturity, and fruit size.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant. A plant cross-pollinates if pollen comes to it from aflower on a different plant.

Corn plants (Zea mays L.) can be bred by both self-pollination andcross-pollination. Both types of pollination involve the corn plant'sflowers. Corn has separate male and female flowers on the same plant,located on the tassel and the ear, respectively. Natural pollinationoccurs in corn when wind blows pollen from the tassels to the silks thatprotrude from the tops of the ear shoot.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants produces a uniform population ofhybrid plants that are heterozygous for many gene loci. Conversely, across of two plants each heterozygous at a number of loci produces apopulation of hybrid plants that differ genetically and are not uniform.The resulting non-uniformity makes performance unpredictable.

The development of uniform corn plant hybrids requires the developmentof homozygous inbred plants, the crossing of these inbred plants, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionare examples of breeding methods used to develop hybrid parent plantsfrom breeding populations. Those breeding methods combine the geneticbackgrounds from two or more inbred plants or various other broad-basedsources into breeding pools from which new inbred plants are developedby selfing and selection of desired phenotypes. The new inbreds arecrossed with other inbred plants and the hybrids from these crosses areevaluated to determine which of those have commercial potential.

North American farmers plant tens of millions of acres of corn at thepresent time and there are extensive national and internationalcommercial corn breeding programs. A continuing goal of these cornbreeding programs is to develop corn hybrids that are based on stableinbred plants and have one or more desirable characteristics. Toaccomplish this goal, the corn breeder must select and develop superiorinbred parental plants.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a corn plant of the hybridvariety designated CH540328. Also provided are corn plants having allthe physiological and morphological characteristics of the hybrid cornvariety CH540328. A hybrid corn plant of the invention may furthercomprise a cytoplasmic or nuclear factor that is capable of conferringmale sterility or otherwise preventing self-pollination, such as byself-incompatibility. Parts of the corn plant of the present inventionare also provided, for example, pollen obtained from a hybrid plant andan ovule of the hybrid plant. The invention also concerns seed of thehybrid corn variety CH540328. The hybrid corn seed of the invention maybe provided as a population of corn seed of the variety designatedCH540328.

In a further aspect, the invention provides a composition comprising aseed of corn variety CH540328 comprised in plant seed growth media. Incertain embodiments, the plant seed growth media is a soil or syntheticcultivation medium. In specific embodiments, the growth medium may becomprised in a container or may, for example, be soil in a field.

In another aspect of the invention, the hybrid corn variety CH540328 isprovided comprising an added desired trait. The desired trait may be agenetic locus that is a dominant or recessive allele. In certainembodiments of the invention, the genetic locus confers traits such as,for example, male sterility, waxy starch, herbicide resistance, insectresistance, resistance to bacterial, fungal, nematode or viral disease,and altered fatty acid, phytate or carbohydrate metabolism. The geneticlocus may be a naturally occurring corn gene introduced into the genomeof a parent of the variety by backcrossing, a natural or inducedmutation, or a transgene introduced through genetic transformationtechniques. When introduced through transformation, a genetic locus maycomprise one or more transgenes integrated at a single chromosomallocation.

In yet another aspect of the invention, a hybrid corn plant of thevariety designated CH540328 is provided, wherein acytoplasmically-inherited trait has been introduced into said hybridplant. Such cytoplasmically-inherited traits are passed to progenythrough the female parent in a particular cross. An exemplarycytoplasmically-inherited trait is the male sterility trait.Cytoplasmic-male sterility (CMS) is a pollen abortion phenomenondetermined by the interaction between the genes in the cytoplasm and thenucleus. Alteration in the mitochondrial genome and the lack of restorergenes in the nucleus will lead to pollen abortion. With either a normalcytoplasm or the presence of restorer gene(s) in the nucleus, the plantwill produce pollen normally. A CMS plant can be pollinated by amaintainer version of the same variety, which has a normal cytoplasm butlacks the restorer gene(s) in the nucleus, and continues to be malesterile in the next generation. The male fertility of a CMS plant can berestored by a restorer version of the same variety, which must have therestorer gene(s) in the nucleus. With the restorer gene(s) in thenucleus, the offspring of the male-sterile plant can produce normalpollen grains and propagate. A cytoplasmically inherited trait may be anaturally occurring maize trait or a trait introduced through genetictransformation techniques.

In another aspect of the invention, a tissue culture of regenerablecells of a plant of variety CH540328 is provided. The tissue culturewill preferably be capable of regenerating plants capable of expressingall of the physiological and morphological characteristics of thevariety, and of regenerating plants having substantially the samegenotype as other plants of the variety. Examples of some of thephysiological and morphological characteristics of the variety CH540328include characteristics related to yield, maturity, and kernel quality,each of which is specifically disclosed herein. The regenerable cells insuch tissue cultures will preferably be derived from embryos,meristematic cells, immature tassels, microspores, pollen, leaves,anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, orstalks, or from callus or protoplasts derived from those tissues. Stillfurther, the present invention provides corn plants regenerated from thetissue cultures of the invention, the plants having all thephysiological and morphological characteristics of variety CH540328.

In still another aspect, the invention provides a method of producinghybrid corn seed comprising crossing a plant of variety CV585594 with aplant of variety CV910316. In a cross, either parent may serve as themale or female. Processes are also provided for producing corn seeds orplants, which processes generally comprise crossing a first parent cornplant with a second parent corn plant, wherein at least one of the firstor second parent corn plants is a plant of the variety designatedCH540328. In such crossing, either parent may serve as the male orfemale parent. These processes may be further exemplified as processesfor preparing hybrid corn seed or plants, wherein a first hybrid cornplant is crossed with a second corn plant of a different, distinctvariety to provide a hybrid that has, as one of its parents, the hybridcorn plant variety CH540328. In these processes, crossing will result inthe production of seed. The seed production occurs regardless of whetherthe seed is collected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting, preferably in pollinating proximity, seeds of afirst and second parent corn plant, and preferably, seeds of a firstcorn plant and a second, distinct corn plant. Where the plants are notin pollinating proximity, pollination can nevertheless be accomplishedby transferring a pollen or tassel bag from one plant to the other asdescribed below.

A second step comprises cultivating or growing the seeds of said firstand second parent corn plants into plants that bear flowers (corn bearsboth male flowers (tassels) and female flowers (silks) in separateanatomical structures on the same plant). A third step comprisespreventing self-pollination of the plants, i.e., preventing the silks ofa plant from being fertilized by any plant of the same variety,including the same plant. This is preferably done by emasculating themale flowers of the first or second parent corn plant, (i.e., treatingor manipulating the flowers so as to prevent pollen production, in orderto produce an emasculated parent corn plant). Self-incompatibilitysystems may also be used in some hybrid crops for the same purpose.Self-incompatible plants still shed viable pollen and can pollinateplants of other varieties but are incapable of pollinating themselves orother plants of the same variety.

A fourth step may comprise allowing cross-pollination to occur betweenthe first and second parent corn plants. When the plants are not inpollinating proximity, this is done by placing a bag, usually paper orglassine, over the tassels of the first plant and another bag over thesilks of the incipient ear on the second plant. The bags are left inplace for at least 24 hours. Since pollen is viable for less than 24hours, this assures that the silks are not pollinated from other pollensources, that any stray pollen on the tassels of the first plant isdead, and that the only pollen transferred comes from the first plant.The pollen bag over the tassel of the first plant is then shakenvigorously to enhance release of pollen from the tassels, and the shootbag is removed from the silks of the incipient ear on the second plant.Finally, the pollen bag is removed from the tassel of the first plantand is placed over the silks of the incipient ear of the second plant,shaken again and left in place. Yet another step comprises harvestingthe seeds from at least one of the parent corn plants. The harvestedseed can be grown to produce a corn plant or hybrid corn plant.

The present invention also provides corn seed and plants produced by aprocess that comprises crossing a first parent corn plant with a secondparent corn plant, wherein at least one of the first or second parentcorn plants is a plant of the variety designated CH540328. In oneembodiment of the invention, corn seed and plants produced by theprocess are first generation hybrid corn seed and plants produced bycrossing an inbred with another, distinct inbred. The present inventionfurther contemplates seed of an F₁ hybrid corn plant. Therefore, certainexemplary embodiments of the invention provide an F₁ hybrid corn plantand seed thereof, specifically the hybrid variety designated CH540328.

Such a plant can be analyzed by its “genetic complement.” This term isused to refer to the aggregate of nucleotide sequences, the expressionof which defines the phenotype of, for example, a corn plant, or a cellor tissue of that plant. A genetic complement thus represents thegenetic make up of an cell, tissue or plant. The invention thus providescorn plant cells that have a genetic complement in accordance with thecorn plant cells disclosed herein, and plants, seeds and diploid plantscontaining such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., marker typing profiles.It is known in the art that such complements may also be identified bymarker types including, but not limited to, Simple Sequence Repeats(SSRs), Simple Sequence Length Polymorphisms (SSLPs) (Williams et al.,Nucleic Acids Res., 18:6531-6535, 1990), Randomly Amplified PolymorphicDNAs (RAPDs), DNA Amplification Fingerprinting (DAF), SequenceCharacterized Amplified Regions (SCARs), Arbitrary Primed PolymeraseChain Reaction (AP-PCR), Amplified Fragment Length Polymorphisms (AFLPs)(EP 0 534 858, specifically incorporated herein by reference in itsentirety), and Single Nucleotide Polymorphisms (SNPs) (Wang et al.,Science, 280:1077-1082, 1998).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions of PlantCharacteristics

Barren Plants: Plants that are barren, i.e., lack an ear with grain, orhave an ear with only a few scattered kernels.

Cg: Colletotrichum graminicola rating. Rating times 10 is approximatelyequal to percent total plant infection.

CLN: Corn Lethal Necrosis (combination of Maize Chlorotic Mottle Virusand Maize Dwarf Mosaic virus) rating: numerical ratings are based on aseverity scale where 1=most resistant to 9=susceptible.

Cn: Corynebacterium nebraskense rating. Rating times 10 is approximatelyequal to percent total plant infection.

Cz: Cercospora zeae-maydis rating. Rating times 10 is approximatelyequal to percent total plant infection.

Dgg: Diatraea grandiosella girdling rating (values are percent plantsgirdled and stalk lodged).

Dropped Ears: Ears that have fallen from the plant to the ground.

Dsp: Diabrotica species root ratings (1=least affected to 9=severepruning).

Ear-Attitude: The attitude or position of the ear at harvest scored asupright, horizontal, or pendant.

Ear-Cob Color: The color of the cob, scored as white, pink, red, orbrown.

Ear-Cob Diameter: The average diameter of the cob measured at themidpoint.

Ear-Cob Strength: A measure of mechanical strength of the cobs tobreakage, scored as strong or weak.

Ear-Diameter: The average diameter of the ear at its midpoint.

Ear-Dry Husk Color: The color of the husks at harvest scored as buff,red, or purple.

Ear-Fresh Husk Color: The color of the husks 1 to 2 weeks afterpollination scored as green, red, or purple.

Ear-Husk Bract: The length of an average husk leaf scored as short,medium, or long.

Ear-Husk Cover: The average distance from the tip of the ear to the tipof the husks, minimum value no less than zero.

Ear-Husk Opening: An evaluation of husk tightness at harvest scored astight, intermediate, or open.

Ear-Length: The average length of the ear.

Ear-Number Per Stalk: The average number of ears per plant.

Ear-Shank Internodes: The average number of internodes on the ear shank.

Ear-Shank Length: The average length of the ear shank.

Ear-Shelling Percent: The average of the shelled grain weight divided bythe sum of the shelled grain weight and cob weight for a single ear.

Ear-Silk Color: The color of the silk observed 2 to 3 days after silkemergence scored as green-yellow, yellow, pink, red, or purple.

Ear-Taper (Shape): The taper or shape of the ear scored as conical,semi-conical, or cylindrical.

Ear-Weight: The average weight of an ear.

Early Stand: The percent of plants that emerge from the ground asdetermined in the early spring.

ER: Ear rot rating (values approximate percent ear rotted).

Final Stand Count: The number of plants just prior to harvest.

GDUs: Growing degree units which are calculated by the Barger Method,where the heat units for a 24-h period are calculated as GDUs=[(Maximumdaily temperature+Minimum daily temperature)/2]−50. The highest maximumdaily temperature used is 86° F. and the lowest minimum temperature usedis 50° F.

GDUs to Shed: The number of growing degree units (GDUs) or heat unitsrequired for a variety to have approximately 50% of the plants sheddingpollen as measured from time of planting. GDUs to shed is determined bysumming the individual GDU daily values from planting date to the dateof 50% pollen shed.

GDUs to Silk: The number of growing degree units for a variety to haveapproximately 50% of the plants with silk emergence as measured fromtime of planting. GDUs to silk is determined by summing the individualGDU daily values from planting date to the date of 50% silking.

Hc2: Helminthosporium carbonum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hc3: Helminthosporium carbonum race 3 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hm: Helminthosporium maydis race 0 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht1: Helminthosporium turcicum race 1 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht2: Helminthosporium turcicum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

HtG: Chlorotic-lesion type resistance. “+” indicates the presence of Htchlorotic-lesion type resistance; “−” indicates absence of Htchlorotic-lesion type resistance; and “+/−” indicates segregation of Htchlorotic-lesion type resistance. Rating times 10 is approximately equalto percent total plant infection.

Kernel-Aleurone Color: The color of the aleurone scored as white, pink,tan, brown, bronze, red, purple, pale purple, colorless, or variegated.

Kernel-Cap Color: The color of the kernel cap observed at dry stage,scored as white, lemon-yellow, yellow, or orange.

Kernel-Endosperm Color: The color of the endosperm scored as white, paleyellow, or yellow.

Kernel-Endosperm Type: The type of endosperm scored as normal, waxy, oropaque.

Kernel-Grade: The percent of kernels that are classified as rounds.

Kernel-Length: The average distance from the cap of the kernel to thepedicel.

Kernel-Number Per Row: The average number of kernels in a single row.

Kernel-Pericarp Color: The color of the pericarp scored as colorless,red-white crown, tan, bronze, brown, light red, cherry red, orvariegated.

Kernel-Row Direction: The direction of the kernel rows on the ear scoredas straight, slightly curved, spiral, or indistinct (scattered).

Kernel-Row Number: The average number of rows of kernels on a singleear.

Kernel-Side Color: The color of the kernel side observed at the drystage, scored as white, pale yellow, yellow, orange, red, or brown.

Kernel-Thickness: The distance across the narrow side of the kernel.

Kernel-Type: The type of kernel scored as dent, flint, or intermediate.

Kernel-Weight: The average weight of a predetermined number of kernels.

Kernel-Width: The distance across the flat side of the kernel.

Kz: Kabatiella zeae rating. Rating times 10 is approximately equal topercent total plant infection.

Leaf-Angle: Angle of the upper leaves to the stalk scored as upright (0to 30 degrees), intermediate (30 to 60 degrees), or lax (60 to 90degrees).

Leaf-Color: The color of the leaves 1 to 2 weeks after pollinationscored as light green, medium green, dark green, or very dark green.

Leaf-Length: The average length of the primary ear leaf.

Leaf-Longitudinal Creases: A rating of the number of longitudinalcreases on the leaf surface 1 to 2 weeks after pollination. Creases arescored as absent, few, or many.

Leaf-Marginal Waves: A rating of the waviness of the leaf margin 1 to 2weeks after pollination, rated as none, few, or many.

Leaf-Number: The average number of leaves of a mature plant. Countingbegins with the cotyledonary leaf and ends with the flag leaf.

Leaf-Sheath Anthocyanin: A rating of the level of anthocyanin in theleaf sheath 1 to 2 weeks after pollination, scored as absent,basal-weak, basal-strong, weak or strong.

Leaf-Sheath Pubescence: A rating of the pubescence of the leaf sheath.Ratings are taken 1 to 2 weeks after pollination and scored as light,medium, or heavy.

Leaf-Width: The average width of the primary ear leaf measured at itswidest point.

LSS: Late season standability (values times 10 approximate percentplants lodged in disease evaluation plots).

Moisture: The moisture of the grain at harvest.

On1: Ostrinia nubilalis 1st brood rating (1=resistant to 9=susceptible).

On2: Ostrinia nubilalis 2nd brood rating (1=resistant to 9=susceptible).

Relative Maturity: A maturity rating based on regression analysis. Theregression analysis is developed by utilizing check hybrids and theirpreviously established day rating versus actual harvest moistures.Harvest moisture on the hybrid in question is determined and thatmoisture value is inserted into the regression equation to yield arelative maturity.

Root Lodging: Root lodging is the percentage of plants that root lodge.A plant is counted as root lodged if a portion of the plant leans fromthe vertical axis by approximately 30 degrees or more.

Seedling Color: Color of leaves at the 6 to 8 leaf stage.

Seedling Height: Plant height at the 6 to 8 leaf stage.

Seedling Vigor: A visual rating of the amount of vegetative growth on a1 to 9 scale, where 1 equals best. The score is taken when the averageentry in a trial is at the fifth leaf stage.

Selection Index: The selection index gives a single measure of hybrid'sworth based on information from multiple traits. One of the traits thatis almost always included is yield. Traits may be weighted according tothe level of importance assigned to them.

Sr: Sphacelotheca reiliana rating is actual percent infection.

Stalk-Anthocyanin: A rating of the amount of anthocyanin pigmentation inthe stalk. The stalk is rated 1 to 2 weeks after pollination as absent,basal-weak, basal-strong, weak, or strong.

Stalk-Brace Root Color: The color of the brace roots observed 1 to 2weeks after pollination as green, red, or purple.

Stalk-Diameter: The average diameter of the lowest visible internode ofthe stalk.

Stalk-Ear Height: The average height of the ear measured from the groundto the point of attachment of the ear shank of the top developed ear tothe stalk.

Stalk-Internode Direction: The direction of the stalk internode observedafter pollination as straight or zigzag.

Stalk-Internode Length: The average length of the internode above theprimary ear.

Stalk Lodging: The percentage of plants that did stalk lodge. Plants arecounted as stalk lodged if the plant is broken over or off below theear.

Stalk-Nodes With Brace Roots: The average number of nodes having braceroots per plant.

Stalk-Plant Height: The average height of the plant as measured from thesoil to the tip of the tassel.

Stalk-Tillers: The percent of plants that have tillers. A tiller isdefined as a secondary shoot that has developed as a tassel capable ofshedding pollen.

Staygreen: Staygreen is a measure of general plant health near the timeof black layer formation (physiological maturity). It is usuallyrecorded at the time the ear husks of most entries within a trial haveturned a mature color. Scoring is on a 1 to 9 basis where 1 equals best.

STR: Stalk rot rating (values represent severity rating of 1=25% ofinoculated internode rotted to 9=entire stalk rotted and collapsed).

SVC: Southeastern Virus Complex (combination of Maize Chlorotic DwarfVirus and Maize Dwarf Mosaic Virus) rating; numerical ratings are basedon a severity scale where 1=most resistant to 9=susceptible.

Tassel-Anther Color: The color of the anthers at 50% pollen shed scoredas green-yellow, yellow, pink, red, or purple.

Tassel-Attitude: The attitude of the tassel after pollination scored asopen or compact.

Tassel-Branch Angle: The angle of an average tassel branch to the mainstem of the tassel scored as upright (less than 30 degrees),intermediate (30 to 45 degrees), or lax (greater than 45 degrees).

Tassel-Branch Number: The average number of primary tassel branches.

Tassel-Glume Band: The closed anthocyanin band at the base of the glumescored as present or absent.

Tassel-Glume Color: The color of the glumes at 50% shed scored as green,red, or purple.

Tassel-Length: The length of the tassel measured from the base of thebottom tassel branch to the tassel tip.

Tassel-Peduncle Length: The average length of the tassel peduncle,measured from the base of the flag leaf to the base of the bottom tasselbranch.

Tassel-Pollen Shed: A visual rating of pollen shed determined by tappingthe tassel and observing the pollen flow of approximately five plantsper entry. Rated on a 1 to 9 scale where 9=sterile, 1=most pollen.

Tassel-Spike Length: The length of the spike measured from the base ofthe top tassel branch to the tassel tip.

Test Weight: Weight of the grain in pounds for a given volume (bushel)adjusted to 15.5% moisture.

Yield: Yield of grain at harvest adjusted to 15.5% moisture.

II. Other Definitions

Allele: Any of one or more alternative forms of a gene locus, all ofwhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny back to one of the parents, for example, a first generationhybrid (F₁) with one of the parental genotypes of the F₁ hybrid.

Crossing: The pollination of a female flower of a corn plant, therebyresulting in the production of seed from the flower.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a chemical agent or a cytoplasmic or nuclear geneticfactor conferring male sterility.

F₁ Hybrid: The first generation progeny of the cross of two plants.

Genetic Complement: An aggregate of nucleotide sequences, the expressionof which sequences defines the phenotype in corn plants, or componentsof plants including cells or tissue.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Genetic loci that contribute, at least inpart, certain numerically representable traits that are usuallycontinuously distributed.

Regeneration: The development of a plant from tissue culture.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing wherein essentially allof the desired morphological and physiological characteristics of aninbred are recovered in addition to the characteristics conferred by thesingle locus transferred into the inbred via the backcrossing technique.A single locus may comprise one gene, or in the case of transgenicplants, one or more transgenes integrated into the host genome at asingle site (locus).

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic sequence which has been introduced into the nuclearor chloroplast genome of a corn plant by a genetic transformationtechnique.

III. Variety Descriptions

In accordance with one aspect of the present invention, there isprovided a novel hybrid corn plant variety designated CH540328. Hybridvariety CH540328 was produced from a cross of the inbred varietiesdesignated CV585594 and CV910316. The inbred parents have beenself-pollinated and ear-rowed a sufficient number of generations withcareful attention paid to uniformity of plant type to show uniformityand stability within the limits of environmental influence.

In accordance with one aspect of the invention, there is provided a cornplant having the physiological and morphological characteristics of cornplant CH540328. An analysis of such morphological traits was carriedout, the results of which are presented in Table 1.

TABLE 1 Morphological Traits for Hybrid Variety CH540328 CHARACTERISTICVALUE 1. STALK Plant Height cm. 269.3  Ear Height cm. 99.3 AnthocyaninAbsent Brace Root Color Absent Internode Direction Straight InternodeLength cm. 18.0 2. LEAF Color Dark Green Length cm. 88.9 Width cm. 11.1Sheath Anthocyanin Weak Sheath Pubescence Very Light Marginal WavesModerate Longitudinal Creases Many 3. TASSEL Length cm. 50.4 PeduncleLength cm. 13.4 Branch Number  6.0 Anther Color Salmon Glume Color GreenGlume Band Absent 4. EAR Silk Color Green-Yellow Number Per Stalk  1.0Length cm. Pendent Position 18.4 Shape Semi-Conical Diameter cm.  5.2Shank Length cm. 12.1 Husk Bract Short Husk Opening  2.8 Husk Cover cm.Tight Husk Color Fresh Green Husk Color Dry Buff Cob Color  2.5 CobDiameter cm. Red Shelling Percent 89.1 5. KERNEL Row Number 17.0 NumberPer Row 40.4 Row Direction Straight Type Dent Cap Color Yellow SideColor Yellow Length (depth) mm. 14.4 Width mm.  8.3 Thickness  4.1Endosperm Type Normal Endosperm Color Yellow *These are typical values.Values may vary due to environment. Other values that are substantiallyequivalent are also within the scope of the invention.

During the development of a hybrid plant detailed evaluations of thephenotype are made including formal comparisons with other commerciallysuccessful hybrids. Because the corn is grown in close proximity,environmental factors that affect gene expression, such as moisture,temperature, sunlight, and pests, are minimized. For a decision to bemade to commercialize a hybrid, it is not necessary that the hybrid bebetter than all other hybrids. Rather, significant improvements must beshown in at least some traits that would create improvements in someniches. Examples of such comparative performance data for the hybridcorn plant CH540328 are set forth herein below in Table 2.

TABLE 2 Comparison of CH540328 With Selected Hybrid Varieties EntriesCompared YLD_BE MST STLP RTLP FNSP SDV PHT EHT TWT STG GSPP GDU SLKCH540328 214.7 22.4 1.2 3.2 98.6 3 95 45 54.9 5.4 0.7 1,219 1,216DKC55-67 199.1 21.5 0.9 1.3 99.4 4 94 43 54.8 6.1 0.8 1,213 1,211Deviation 15.59 0.9 0.35 1.83 −0.81 −1.04 1.29 1.71 0.13 −0.78 −0.036.13 4.97 Significance ** ** * + ** ** Significance levels are indicatedas: + = 10%, * = 5%, ** = 1% LEGEND ABBREVIATIONS: YLD_B = Yield(bushels/acre) MST = Moisture STLP = Stalk Lodging (percent) RTLP = RootLodging (percent) FNSP = Final Stand (percent of test mean) SDV =Seedling Vigor Rating PHT = Plant Height (inches) EHT = Ear Height(inches) TWT = Test Weight (pounds) STG = Staygreen Rating GSPP =Greensnap (percentage) GDU = GDUs to Shed SLK = GDUs to Silk

In accordance with another aspect of the present invention, there isprovided a corn plant having the physiological and morphologicalcharacteristics of corn plant CV910316. A description of thephysiological and morphological characteristics of corn plant CV910316is presented in Table 3.

TABLE 3 Morphological Traits for Corn Variety CV910316 CHARACTERISTICVALUE 1. STALK Plant Height (cm.) 192.4  Ear Height (cm) 77.9Anthocyanin Absent Brace Root Color Absent Internode Direction StraightInternode Length cm. 14.6 2. LEAF Color Green Length cm. 69.7 Width cm. 9.3 Sheath Anthocyanin Weak Sheath Pubescence None Marginal Waves FewLongitudinal Creases Many 3. TASSEL Length cm. 43.0 Peduncle Length cm. 9.7 Branch Number  4.0 Anther Color Salmon Glume Color Green Glume BandAbsent 4. EAR Silk Color Green-Yellow Number Per Stalk  1.0 Position(attitude) Upright Length cm. 13.3 Shape Semi-Conical Diameter cm.  4.7Shank Length cm.  6.8 Husk Bract Short Husk Cover cm.  6.5 Husk OpeningTight Husk Color Fresh Green Husk Color Dry Buff Cob Diameter cm.  2.6Cob Color Red Shelling Percent 87.6 5. KERNEL Row Number 18.0 Number PerRow 26.8 Row Direction Straight Type Dent Cap Color Yellow Side ColorYellow Length (depth) mm. 11.6 Width mm.  7.8 Thickness  4.5 EndospermType Normal Endosperm Color Yellow *These are typical values. Values mayvary due to environment. Other values that are substantially equivalentare also within the scope of the invention.

IV. Deposit Information

A deposit of at least 2500 seeds of inbred parent plant varietiesCV585594 (U.S. Pat. No. 8,076,550) and CV910316 (U.S. patent applicationSer. No. 13/096,713, filed Apr. 28, 2011, which issued as U.S. Pat. No.8,507,774) has been made with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA, andassigned ATCC Accession Nos. PTA-10673, and PTA-11923, respectively. Theseeds were deposited with the ATCC on Feb. 18, 2010, and Jun. 7, 2011,respectively. Access to the deposits will be available during thependency of the application to the Commissioner of Patents andTrademarks and persons determined by the Commissioner to be entitledthereto upon request. The deposits will be maintained in the ATCCDepository, which is a public depository, for a period of 30 years, or 5years after the most recent request, or for the enforceable life of thepatent, whichever is longer, and will be replaced if it becomesnonviable during that period. Applicant does not waive any infringementof their rights granted under this patent or under the Plant VarietyProtection Act (7 U.S.C. 2321 et seq.).

V. Further Embodiments of the Invention

In one embodiment, compositions are provided comprising a seed of cornvariety CH540328 comprised in plant seed cultivation media. Plant seedcultivation media are well known to those of skill in the art andinclude, but are in no way limited to, soil or synthetic cultivationmedium. Advantageously, plant seed cultivation media can provideadequate physical support for seeds and can retain moisture and/ornutritional components. Examples of characteristics for soils that maybe desirable in certain embodiments can be found, for instance, in U.S.Pat. Nos. 3,932,166 and 4,707,176. Synthetic plant cultivation media arealso well known in the art and may, in certain embodiments, comprisepolymers or hydrogels. Examples of such compositions are described, forexample, in U.S. Pat. No. 4,241,537.

In certain further aspects, the invention provides plants modified toinclude at least a first desired trait. Such plants may, in oneembodiment, be developed by a plant breeding technique calledbackcrossing, wherein essentially all of the morphological andphysiological characteristics of a variety are recovered in addition toa genetic locus transferred into the hybrid via the backcrossingtechnique. By essentially all of the morphological and physiologicalcharacteristics, it is meant that all of the characteristics of a plantare recovered that are otherwise present when compared in the sameenvironment, other than an occasional variant trait that might ariseduring backcrossing or direct introduction of a transgene. In oneembodiment, such traits may be determined, for example, relative to thetraits listed in Table 1 as determined at the 5% significance level whengrown under the same environmental conditions.

Backcrossing methods can be used with the present invention to improveor introduce a trait in a hybrid via modification of its inbredparent(s). The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental corn plants forthat hybrid. The parental corn plant which contributes the locus or locifor the desired trait is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur.

The parental corn plant to which the locus or loci from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman et al., In:Breeding Field Crops, 4th Ed., Iowa State University Press, Ames, Iowa,pp 132-155 and 321-344, 1995; Fehr, In: Principles of CultivarDevelopment, 1:360-376, 1987; Sprague and Dudley (eds.), In: Corn andCorn Improvement, 3^(rd) Ed., Crop Science of America, Inc., and SoilScience of America, Inc., Madison Wis. 881-883; 901-918, 1988). In atypical backcross protocol, the original parent hybrid of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the genetic locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a corn plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the transferred locus from thenonrecurrent parent. The backcross process may be accelerated by the useof genetic markers, such as SSR, RFLP, SNP or AFLP markers to identifyplants with the greatest genetic complement from the recurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto add or substitute one or more new traits in the original inbred andhybrid progeny therefrom. To accomplish this, a genetic locus of therecurrent parent is modified or substituted with the desired locus fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological constitution of the original plant. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many traits have been identified that are not regularly selected for inthe development of a new variety but that can be improved bybackcrossing techniques. A genetic locus conferring the traits may ormay not be transgenic. Examples of such traits known to those of skillin the art include, but are not limited to, male sterility, waxy starch,herbicide resistance, resistance for bacterial, fungal, or viraldisease, insect resistance, male fertility and enhanced nutritionalquality. These genes are generally inherited through the nucleus, butmay be inherited through the cytoplasm. Some known exceptions to thisare genes for male sterility, some of which are inheritedcytoplasmically, but still act as a single locus trait.

Direct selection may be applied where a genetic locus acts as a dominanttrait. An example of a dominant trait is the herbicide resistance trait.For this selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants which do not have the desired herbicide resistancecharacteristic, and only those plants which have the herbicideresistance gene are used in the subsequent backcross. This process isthen repeated for all additional backcross generations.

Many useful traits are those which are introduced by genetictransformation techniques. Methods for the genetic transformation ofcorn are known to those of skill in the art. For example, methods whichhave been described for the genetic transformation of corn includeelectroporation (U.S. Pat. No. 5,384,253), electrotransformation (U.S.Pat. No. 5,371,003), microprojectile bombardment (U.S. Pat. Nos.5,550,318, 5,736,369 and 5,538,880; and PCT Publication WO 95/06128),Agrobacterium-mediated transformation (U.S. Pat. No. 5,591,616 and E.P.Publication EP672752), direct DNA uptake transformation of protoplasts(Omirulleh et al., Plant Mol. Biol., 21(3):415-428, 1993) and siliconcarbide fiber-mediated transformation (U.S. Pat. Nos. 5,302,532 and5,464,765).

It is understood to those of skill in the art that a transgene need notbe directly transformed into a plant, as techniques for the productionof stably transformed corn plants that pass single loci to progeny byMendelian inheritance is well known in the art. Such loci may thereforebe passed from parent plant to progeny plants by standard plant breedingtechniques that are well known in the art.

A. Male Sterility

Examples of genes conferring male sterility include those disclosed inU.S. Pat. Nos. 3,861,709, 3,710,511, 4,654,465, 5,625,132, and4,727,219, each of the disclosures of which are specificallyincorporated herein by reference in their entirety. Male sterility genescan increase the efficiency with which hybrids are made, in that theyeliminate the need to physically emasculate the corn plant used as afemale in a given cross.

Where one desires to employ male-sterility systems with a corn plant inaccordance with the invention, it may be beneficial to also utilize oneor more male-fertility restorer genes. For example, where cytoplasmicmale sterility (CMS) is used, hybrid seed production requires threeinbred lines: (1) a cytoplasmically male-sterile line having a CMScytoplasm; (2) a fertile inbred with normal cytoplasm, which is isogenicwith the CMS line for nuclear genes (“maintainer line”); and (3) adistinct, fertile inbred with normal cytoplasm, carrying a fertilityrestoring gene (“restorer” line). The CMS line is propagated bypollination with the maintainer line, with all of the progeny being malesterile, as the CMS cytoplasm is derived from the female parent. Thesemale sterile plants can then be efficiently employed as the femaleparent in hybrid crosses with the restorer line, without the need forphysical emasculation of the male reproductive parts of the femaleparent.

The presence of a male-fertility restorer gene results in the productionof fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the corn plant is utilized, e.g.,for silage, but in most cases, the seeds will be deemed the mostvaluable portion of the crop, so fertility of the hybrids in these cropsmust be restored. Therefore, one aspect of the current inventionconcerns the hybrid corn plant CH540328 comprising a genetic locuscapable of restoring male fertility in an otherwise male-sterile plant.Examples of male-sterility genes and corresponding restorers which couldbe employed with the plants of the invention are well known to those ofskill in the art of plant breeding and are disclosed in, for instance,U.S. Pat. Nos. 5,530,191; 5,689,041; 5,741,684; and 5,684,242, thedisclosures of which are each specifically incorporated herein byreference in their entirety.

B. Herbicide Resistance

Numerous herbicide resistance genes are known and may be employed withthe invention. An example is a gene conferring resistance to a herbicidethat inhibits the growing point or meristem, such as an imidazalinone ora sulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee et al., EMBO J., 7:1241,1988; Gleen et al., Plant Molec. Biology, 18:1185-1187, 1992; and Mikiet al., Theor. Appl. Genet., 80:449, 1990.

Resistance genes for glyphosate (resistance conferred by mutant5-enolpyruvl-3 phosphikimate synthase (EPSP) and aroA genes,respectively), and hygromycin B phosphotransferase, and to otherphosphono compounds such as glufosinate (phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicus phosphinothricin-acetyltransferase (bar) genes) may also be used. See, for example, U.S. Pat.No. 4,940,835 to Shah, et al., which discloses the nucleotide sequenceof a form of EPSPS which can confer glyphosate resistance. A DNAmolecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. A hygromycin Bphosphotransferase gene from E. coli that confers resistance toglyphosate in tobacco callus and plants is described in Penaloza-Vazquezet al. (Plant Cell Reports, 14:482-487, 1995). European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyltransferase gene isprovided in European application No. 0 242 246 to Leemans et al. DeGreefet al., (Biotechnology, 7:61, 1989), describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary of genesconferring resistance to phenoxy propionic acids and cycloshexones, suchas sethoxydim and haloxyfop are the Acct-S1, Accl-S2 and Acct-S3 genesdescribed by Marshall et al., (Theor. Appl. Genet., 83:4:35, 1992).

Genes conferring resistance to a herbicide that inhibits photosynthesisare also known, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla et al., (Plant Cell, 3:169,1991), describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA moleculescontaining these genes are available under ATCC Accession Nos. 53435,67441, and 67442. Cloning and expression of DNA coding for a glutathioneS-transferase is described by Hayes et al., (Biochem. J., 285(Pt1):173-180, 1992). Protoporphyrinogen oxidase (PPO) is the target of thePPO-inhibitor class of herbicides; a PPO-inhibitor resistant PPO genewas recently identified in Amaranthus tuberculatus (Patzoldt et al.,PNAS, 103(33):12329-2334, 2006). The herbicide methyl viologen inhibitsCO₂ assimilation. Foyer et al. (Plant Physiol., 109:1047-1057, 1995)describe a plant overexpressing glutathione reductase (GR) which isresistant to methyl viologen treatment.

Siminszky (Phytochemistry Reviews, 5:445-458, 2006) describes plantcytochrome P450-mediated detoxification of multiple, chemicallyunrelated classes of herbicides.

C. Waxy Starch

The waxy characteristic is an example of a recessive trait. In thisexample, the progeny resulting from the first backcross generation (BC1)must be grown and selfed. A test is then run on the selfed seed from theBC1 plant to determine which BC1 plants carried the recessive gene forthe waxy trait. In other recessive traits additional progeny testing,for example growing additional generations such as the BC1S1, may berequired to determine which plants carry the recessive gene.

D. Disease Resistance

Plant defenses are often activated by specific interaction between theproduct of a disease resistance gene (R) in the plant and the product ofa corresponding avirulence (Avr) gene in the pathogen. A plant line canbe transformed with a cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example, Jones et al.,Science, 266:7891, 1994 (cloning of the tomato Cf-9 gene for resistanceto Cladosporium falvum); Martin et al., Science, 262: 1432, 1993 (tomatoPto gene for resistance to Pseudomonas syringae pv.); and Mindrinos etal., Cell, 78(6):1089-1099, 1994 (Arabidopsis RPS2 gene for resistanceto Pseudomonas syringae).

A viral-invasive protein or a complex toxin derived therefrom may alsobe used for viral disease resistance. For example, the accumulation ofviral coat proteins in transformed plant cells imparts resistance toviral infection and/or disease development effected by the virus fromwhich the coat protein gene is derived, as well as by related viruses.See Beachy et al., (Ann. Rev. Phytopathol., 28:451, 1990). Coatprotein-mediated resistance has been conferred upon transformed plantsagainst alfalfa mosaic virus, cucumber mosaic virus, tobacco streakvirus, potato virus X, potato virus Y, tobacco etch virus, tobaccorattle virus and tobacco mosaic virus. Id.

A virus-specific antibody may also be used. See, for example,Tavladoraki et al., (Nature, 366:469, 1993), who show that transgenicplants expressing recombinant antibody genes are protected from virusattack. Additional means of inducing whole-plant resistance to apathogen include modulation of the systemic acquired resistance (SAR) orpathogenesis related (PR) genes, for example genes homologous to theArabidopsis thaliana NIM1/NPR1/SAI1, and/or by increasing salicylic acidproduction (Ryals et al., Plant Cell, 8:1809-1819, 1996).

Logemann et al., (Biotechnology, 10:305, 1992), for example, disclosetransgenic plants expressing a barley ribosome-inactivating gene have anincreased resistance to fungal disease. Plant defensins may be used toprovide resistance to fungal pathogens (Thomma et al., Planta,216:193-202, 2002). Other examples of fungal disease resistance areprovided in U.S. Pat. Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407;6,215,048; 5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,506,962.

E. Insect Resistance

One example of an insect resistance gene includes a Bacillusthuringiensis (Bt) protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., (Gene,48:109-118, 1986), who disclose the cloning and nucleotide sequence of aBt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genescan be purchased from the American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998. Another example is a lectin. See, for example, Van Damme et al.,(Plant Molec. Biol., 24:25, 1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes. Avitamin-binding protein may also be used, such as avidin. See PCTapplication US93/06487, the contents of which are hereby incorporated byreference. This application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

Yet another insect resistance gene is an enzyme inhibitor, for example,a protease or proteinase inhibitor or an amylase inhibitor. See, forexample, Abe et al., (J. Biol. Chem., 262:16793, 1987) (nucleotidesequence of rice cysteine proteinase inhibitor), Huub et al., (PlantMolec. Biol., 21:985, 1993) (nucleotide sequence of cDNA encodingtobacco proteinase inhibitor I), and Sumitani et al., (Biosci. Biotech.Biochem., 57:1243, 1993) (nucleotide sequence of Streptomycesnitrosporeus α-amylase inhibitor).

An insect-specific hormone or pheromone may also be used. See, forexample, the disclosure by Hammock et al., (Nature, 344:458, 1990), ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone, Gade and Goldsworthy (Eds.Physiological System in Insects, Elsevier Academic Press, Burlington,Mass., 2007), describing allostatins and their potential use in pestcontrol; and Palli et al. (Vitam. Horm., 73:59-100, 2005), disclosinguse of ecdysteroid and ecdysteroid receptor in agriculture. The diuretichormone receptor (DHR) was identified in Price et al. (Insect Mol.Biol., 13:469-480, 2004) as a candidate target of insecticides.

Still other examples include an insect-specific antibody or animmunotoxin derived therefrom and a developmental-arrestive protein. SeeTaylor et al., (Seventh Int'l Symposium on Molecular Plant-MicrobeInteractions, Edinburgh, Scotland, Abstract W97, 1994), who describedenzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments.

Nematode resistance has been described, for example, in U.S. Pat. No.6,228,992 and bacterial disease resistance in U.S. Pat. No. 5,516,671.

F. Modified Fatty Acid, Phytate and Carbohydrate Metabolism

Genes may be used conferring modified fatty acid metabolism. Forexample, stearyl-ACP desaturase genes may be used. See Knutzon et al.,(Proc. Natl. Acad. Sci. USA, 89:2624, 1992). Various fatty aciddesaturases have also been described, such as a Saccharomyces cerevisiaeOLE1 gene encoding Δ9 fatty acid desaturase, an enzyme which forms themonounsaturated palmitoleic (16:1) and oleic (18:1) fatty acids frompalmitoyl (16:0) or stearoyl (18:0) CoA (McDonough et al., J. Biol.Chem., 267(9):5931-5936, 1992); a gene encoding a stearoyl-acyl carrierprotein delta-9 desaturase from castor (Fox et al. Proc. Natl. Acad.Sci. USA, 90(6):2486-2490, 1993); Δ6- and Δ12-desaturases from thecyanobacteria Synechocystis responsible for the conversion of linoleicacid (18:2) to gamma-linolenic acid (18:3 gamma) (Reddy et al., PlantMol. Biol., 22(2):293-300, 1993); a gene from Arabidopsis thaliana thatencodes an omega-3 desaturase (Arondel et al., Science,258(5086):1353-1355 1992); plant Δ9-desaturases (PCT Application Publ.No. WO 91/13972) and soybean and Brassica Δ15 desaturases (EuropeanPatent Application Publ. No. EP 0616644).

Phytate metabolism may also be modified by introduction of aphytase-encoding gene to enhance breakdown of phytate, adding more freephosphate to the transformed plant. For example, see Van Hartingsveldtet al., (Gene, 127:87, 1993), for a disclosure of the nucleotidesequence of an Aspergillus niger phytase gene. In corn, this, forexample, could be accomplished by cloning and then reintroducing DNAassociated with the single allele which is responsible for corn mutantscharacterized by low levels of phytic acid. See Raboy et al., PlantPhysiol., 124(1):355-368, 1990.

A number of genes are known that may be used to alter carbohydratemetabolism. For example, plants may be transformed with a gene codingfor an enzyme that alters the branching pattern of starch. See Shirozaet al., (J. Bacteriol., 170:810, 1988) (nucleotide sequence ofStreptococcus mutans fructosyltransferase gene), Steinmetz et al., (Mol.Gen. Genet., 20:220, 1985) (nucleotide sequence of Bacillus subtilislevansucrase gene), Pen et al., (Biotechnology, 10:292, 1992)(production of transgenic plants that express Bacillus licheniformisα-amylase), Elliot et al., (Plant Molec. Biol., 21:515, 1993)(nucleotide sequences of tomato invertase genes), Sergaard et al., (J.Biol. Chem., 268:22480, 1993) (site-directed mutagenesis of barleyα-amylase gene), and Fisher et al., (Plant Physiol., 102:1045, 1993)(maize endosperm starch branching enzyme II). The Z10 gene encoding a 10kD zein storage protein from maize may also be used to alter thequantities of 10 kD zein in the cells relative to other components(Kirihara et al., Gene, 71(2):359-370, 1988).

U.S. Patent Appl. Pub. No. 20030163838 describes maize cellulosesynthase genes and methods of use thereof.

G. Resistance to Abiotic Stress

Abiotic stress includes dehydration or other osmotic stress, salinity,high or low light intensity, high or low temperatures, submergence,exposure to heavy metals, and oxidative stress.Delta-pyrroline-5-carboxylate synthetase (P5CS) from mothbean has beenused to provide protection against general osmotic stress.Mannitol-1-phosphate dehydrogenase (mt1D) from E. coli has been used toprovide protection against drought and salinity. Choline oxidase (codAfrom Arthrobactor globiformis) can protect against cold and salt. E.coli choline dehydrogenase (betA) provides protection against salt.Additional protection from cold can be provided by omega-3-fatty aciddesaturase (fad7) from Arabidopsis thaliana. Trehalose-6-phosphatesynthase and levan sucrase (SacB) from yeast and Bacillus subtilis,respectively, can provide protection against drought (summarized fromAnnex II Genetic Engineering for Abiotic Stress Tolerance in Plants,Consultative Group On International Agricultural Research TechnicalAdvisory Committee). Overexpression of superoxide dismutase can be usedto protect against superoxides, as described in U.S. Pat. No. 5,538,878to Thomas et al.

H. Additional Traits

Additional traits can be introduced into the corn variety of the presentinvention. A non-limiting example of such a trait is a coding sequencewhich decreases RNA and/or protein levels. The decreased RNA and/orprotein levels may be achieved through RNAi methods, such as thosedescribed in U.S. Pat. No. 6,506,559 to Fire and Mellow.

Another trait that may find use with the corn variety of the inventionis a sequence which allows for site-specific recombination. Examples ofsuch sequences include the FRT sequence, used with the FLP recombinase(Zhu and Sadowski, J. Biol. Chem., 270:23044-23054, 1995); and the LOXsequence, used with CRE recombinase (Sauer, Mol. Cell. Biol.,7:2087-2096, 1987). The recombinase genes can be encoded at any locationwithin the genome of the corn plant, and are active in the hemizygousstate.

It may also be desirable to make corn plants more tolerant to or moreeasily transformed with Agrobacterium tumefaciens. Expression of p53 andiap, two baculovirus cell-death suppressor genes, inhibited tissuenecrosis and DNA cleavage. Additional targets can include plant-encodedproteins that interact with the Agrobacterium Vir genes; enzymesinvolved in plant cell wall formation; and histones, histoneacetyltransferases and histone deacetylases (reviewed in Gelvin,Microbiology & Mol. Biol. Reviews, 67:16-37, 2003).

In addition to the modification of oil, fatty acid or phytate contentdescribed above, it may additionally be beneficial to modify the amountsor levels of other compounds. For example, the amount or composition ofantioxidants can be altered. See, for example, U.S. Pat. No. 6,787,618,U.S. Patent Appl. Pub. No. 20040034886 and International Patent Appl.Pub. No. WO 00/68393, which disclose the manipulation of antioxidantlevels, and International Patent Appl. Pub. No. WO 03/082899, whichdiscloses the manipulation of a antioxidant biosynthetic pathway.

Additionally, seed amino acid content may be manipulated. U.S. Pat. No.5,850,016 and International Patent Appl. Pub. No. WO 99/40209 disclosethe alteration of the amino acid compositions of seeds. U.S. Pat. Nos.6,080,913 and 6,127,600 disclose methods of increasing accumulation ofessential amino acids in seeds.

U.S. Pat. No. 5,559,223 describes synthetic storage proteins in whichthe levels of essential amino acids can be manipulated. InternationalPatent Appl. Pub. No. WO 99/29882 discloses methods for altering aminoacid content of proteins. International Patent Appl. Pub. No. WO98/20133 describes proteins with enhanced levels of essential aminoacids. International Patent Appl. Pub. No. WO 98/56935 and U.S. Pat.Nos. 6,346,403, 6,441,274 and 6,664,445 disclose plant amino acidbiosynthetic enzymes. International Patent Appl. Pub. No. WO 98/45458describes synthetic seed proteins having a higher percentage ofessential amino acids than wild-type.

U.S. Pat. No. 5,633,436 discloses plants comprising a higher content ofsulfur-containing amino acids; U.S. Pat. No. 5,885,801 discloses plantscomprising a high threonine content; U.S. Pat. No. 5,885,802 disclosesplants comprising a high methionine content; U.S. Pat. No. 5,912,414discloses plants comprising a high methionine content; U.S. Pat. No.5,990,389 discloses plants comprising a high lysine content; U.S. Pat.No. 6,459,019 discloses plants comprising an increased lysine andthreonine content; International Patent Appl. Pub. No. WO 98/42831discloses plants comprising a high lysine content; International PatentAppl. Pub. No. WO 96/01905 discloses plants comprising a high threoninecontent; and International Patent Appl. Pub. No. WO 95/15392 disclosesplants comprising a high lysine content.

I. Origin and Breeding History of an Exemplary Introduced Trait

Provided by the invention are a hybrid plant in which one or more of theparents comprise an introduced trait. Such a plant may be defined ascomprising a single locus conversion. Exemplary procedures for thepreparation of such single locus conversions are disclosed in U.S. Pat.No. 7,205,460, the entire disclosure of which is specificallyincorporated herein by reference.

An example of a single locus conversion is 85DGD1. 85DGD1 MLms is aconversion of 85DGD1 to cytoplasmic male sterility. 85DGD1 MLms wasderived using backcross methods. 85DGD1 (a proprietary inbred ofMonsanto Company) was used as the recurrent parent and MLms, a germplasmsource carrying ML cytoplasmic sterility, was used as the nonrecurrentparent. The breeding history of the converted inbred 85DGD1 MLms can besummarized as follows:

Hawaii Nurseries Planting Made up S-O: Female row 585 male Date Apr. 02,1992 row 500 Hawaii Nurseries Planting S-O was grown and plants wereDate Jul. 15, 1992 backcrossed times 85DGD1 (rows 444 {acute over ( )}443) Hawaii Nurseries Planting Bulked seed of the BC1 was grown and DateNov. 18, 1992 backcrossed times 85DGD1 (rows V3-27 {acute over ( )}V3-26) Hawaii Nurseries Planting Bulked seed of the BC2 was grown andDate Apr. 02, 1993 backcrossed times 85DGD1 (rows 37 {acute over ( )}36) Hawaii Nurseries Planting Bulked seed of the BC3 was grown and DateJul. 14, 1993 backcrossed times 85DGD1 (rows 99 {acute over ( )} 98)Hawaii Nurseries Planting Bulked seed of BC4 was grown and Date Oct. 28,1993 backcrossed times 85DGD1 (rows KS-63 {acute over ( )} KS-62) Summer1994 A single ear of the BC5 was grown and backcrossed times 85DGD1(MC94-822 {acute over ( )} MC94-822-7) Winter 1994 Bulked seed of theBC6 was grown and backcrossed times 85DGD1 (3Q-1 {acute over ( )} 3Q-2)Summer 1995 Seed of the BC7 was bulked and named 85DGD1 MLms.

As described, techniques for the production of corn plants with addedtraits are well known in the art (see, e.g., Poehlman et al., In:Breeding Field Crops, 4th Ed., Iowa State University Press, Ames, Iowa,pp 132-155 and 321-344, 1995; Fehr, In: Principles of CultivarDevelopment, 1:360-376, 1987; Sprague and Dudley (eds.), In: Corn andCorn Improvement, 3^(rd) Ed., Crop Science of America, Inc., and SoilScience of America, Inc., Madison Wis. 881-883; 901-918, 1988). Anon-limiting example of such a procedure one of skill in the art coulduse for preparation of a hybrid corn plant CH540328 comprising an addedtrait is as follows:

-   -   (a) crossing a parent of hybrid corn plant CH540328 such as        CV585594 and/or CV910316 to a second (nonrecurrent) corn plant        comprising a locus to be converted in the parent;    -   (b) selecting at least a first progeny plant resulting from the        crossing and comprising the locus;    -   (c) crossing the selected progeny to the parent line of corn        plant CH540328;    -   (d) repeating steps (b) and (c) until a parent line of variety        CH540328 is obtained comprising the locus; and    -   (e) crossing the converted parent with the second parent to        produce hybrid variety CH540328 comprising a desired trait.

Following these steps, essentially any locus may be introduced intohybrid corn variety CH540328. For example, molecular techniques allowintroduction of any given locus, without the need for phenotypicscreening of progeny during the backcrossing steps.

PCR and Southern hybridization are two examples of molecular techniquesthat may be used for confirmation of the presence of a given locus andthus conversion of that locus. The techniques are carried out asfollows: Seeds of progeny plants are grown and DNA isolated from leaftissue (see Sambrook et al., In: Molecular cloning, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001; Shure et al., Cell,35(1):225-233, 1983). Approximately one gram of leaf tissue islyophilized overnight in 15 ml polypropylene tubes. Freeze-dried tissueis ground to a powder in the tube using a glass rod. Powdered tissue ismixed thoroughly with 3 ml extraction buffer (7.0 M urea, 0.35 M NaCl,0.05 M Tris-HCI pH 8.0, 0.01 M EDTA, 1% sarcosine). Tissue/bufferhomogenate is extracted with 3 ml phenol/chloroform. The aqueous phaseis separated by centrifugation, and precipitated twice using 1/10 volumeof 4.4 M ammonium acetate pH 5.2, and an equal volume of isopropanol.The precipitate is washed with 75% ethanol and resuspended in 100-500 μlTE (0.01 M Tris-HCI, 0.001 M EDTA, pH 8.0). The DNA may then be screenedas desired for presence of the locus.

For PCR, 200-1000 ng genomic DNA from the progeny plant being screenedis added to a reaction mix containing 10 mM Tris-HCl pH 8.3, 1.5 mMMgCl₂, 50 mM KCl, 0.1 mg/ml gelatin, 200 μM each dATP, dCTP, dGTP, dTTP,20% glycerol, 2.5 units Taq DNA polymerase and 0.5 μM each of forwardand reverse DNA primers that span a segment of the locus beingconverted. The reaction is run in a thermal cycling machine 3 minutes at94 C, 39 repeats of the cycle 1 minute at 94 C, 1 minute at 50 C, 30seconds at 72 C, followed by 5 minutes at 72 C. Twenty μl of eachreaction mix is run on a 3.5% NuSieve gel in TBE buffer (90 mMTris-borate, 2 mM EDTA) at 50V for two to four hours. The amplifiedfragment is detected using an agarose gel. Detection of an amplifiedfragment corresponding to the segment of the locus spanned by theprimers indicates the presence of the locus.

For Southern analysis, plant DNA is restricted, separated in an agarosegel and transferred to a Nylon filter in 10×SCP (20 SCP: 2M NaCl, 0.6 Mdisodium phosphate, 0.02 M disodium EDTA) according to standard methods(Southern, J. Mol. Biol., 98:503-517, 1975). Locus DNA or RNA sequencesare labeled, for example, radioactively with ³²P by random priming(Feinberg & Vogelstein, Anal. Biochem., 132(1):6-13, 1983). Filters areprehybridized in 6×SCP, 10% dextran sulfate, 2% sarcosine, and 500 μg/mldenatured salmon sperm DNA. The labeled probe is denatured, hybridizedto the filter and washed in 2×SCP, 1% SDS at 65° C. for 30 minutes andvisualized by autoradiography using Kodak XAR5 film. Presence of thelocus is indicated by detection of restriction fragments of theappropriate size.

VI. Tissue Cultures and In Vitro Regeneration of Corn Plants

A further aspect of the invention relates to tissue cultures of the cornplant designated CH540328. As used herein, the term “tissue culture”indicates a composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant. Exemplary types of tissue cultures are protoplasts, calli andplant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, anthers, silk, and the like. In a preferredembodiment, the tissue culture comprises embryos, protoplasts,meristematic cells, pollen, leaves or anthers derived from immaturetissues of these plant parts. Means for preparing and maintaining planttissue cultures are well known in the art (U.S. Pat. Nos. 5,538,880 and5,550,318, each incorporated herein by reference in their entirety). Byway of example, a tissue culture comprising organs such as tassels oranthers has been used to produce regenerated plants (U.S. Pat. Nos.5,445,961 and 5,322,789; the disclosures of which are incorporatedherein by reference).

One type of tissue culture is tassel/anther culture. Tassels containanthers which in turn enclose microspores. Microspores develop intopollen. For anther/microspore culture, if tassels are the plantcomposition, they are preferably selected at a stage when themicrospores are uninucleate, that is, include only one, rather than 2 or3 nuclei. Methods to determine the correct stage are well known to thoseskilled in the art and include mitramycin fluorescent staining (Pace etal., Theoretical and Applied Genetics, 73:863-869, 1987), trypan blue(preferred) and acetocarmine squashing. The mid-uninucleate microsporestage has been found to be the developmental stage most responsive tothe subsequent methods disclosed to ultimately produce plants.

Although microspore-containing plant organs such as tassels cangenerally be pretreated at any cold temperature below about 25° C., arange of 4 to 25° C. is preferred, and a range of 8 to 14° C. isparticularly preferred. Although other temperatures yield embryoids andregenerated plants, cold temperatures produce optimum response ratescompared to pretreatment at temperatures outside the preferred range.Response rate is measured as either the number of embryoids or thenumber of regenerated plants per number of microspores initiated inculture. Exemplary methods of microspore culture are disclosed in, forexample, U.S. Pat. Nos. 5,322,789 and 5,445,961, the disclosures ofwhich are specifically incorporated herein by reference.

Although not required, when tassels are employed as the plant organ, itis generally preferred to sterilize their surface. Following surfacesterilization of the tassels, for example, with a solution of calciumhypochloride, the anthers are removed from about 70 to 150 spikelets(small portions of the tassels) and placed in a preculture orpretreatment medium. Larger or smaller amounts can be used depending onthe number of anthers.

When one elects to employ tassels directly, tassels are preferablypretreated at a cold temperature for a predefined time, preferably at10° C. for about 4 days. After pretreatment of a whole tassel at a coldtemperature, dissected anthers are further pretreated in an environmentthat diverts microspores from their developmental pathway. The functionof the preculture medium is to switch the developmental program from oneof pollen development to that of embryoid/callus development. Anembodiment of such an environment in the form of a preculture mediumincludes a sugar alcohol, for example mannitol or sorbitol, inositol orthe like. An exemplary synergistic combination is the use of mannitol ata temperature of about 10° C. for a period ranging from about 10 to 14days. In a preferred embodiment, 3 ml of 0.3 M mannitol combined with 50mg/l of ascorbic acid, silver nitrate, and colchicine is used forincubation of anthers at 10° C. for between 10 and 14 days. Anotherembodiment is to substitute sorbitol for mannitol. The colchicineproduces chromosome doubling at this early stage. The chromosomedoubling agent is preferably only present at the preculture stage.

It is believed that the mannitol or other similar carbon structure orenvironmental stress induces starvation and functions to forcemicrospores to focus their energies on entering developmental stages.The cells are unable to use, for example, mannitol as a carbon source atthis stage. It is believed that these treatments confuse the cellscausing them to develop as embryoids and plants from microspores.Dramatic increases in development from these haploid cells, as high as25 embryoids in 10⁴ microspores, have resulted from using these methods.

To isolate microspores, an isolation media is preferred. An isolationmedia is used to separate microspores from the anther walls whilemaintaining their viability and embryogenic potential. An illustrativeembodiment of an isolation media includes a 6% sucrose or maltosesolution combined with an antioxidant such as 50 mg/l of ascorbic acid,0.1 mg/l biotin, and 400 mg/l of proline, combined with 10 mg/l ofnicotinic acid and 0.5 mg/l AgNO₃. In another embodiment, the biotin andproline are omitted.

An isolation media preferably has a higher antioxidant level where it isused to isolate microspores from a donor plant (a plant from which aplant composition containing a microspore is obtained) that is fieldgrown in contrast to greenhouse grown. A preferred level of ascorbicacid in an isolation medium is from about 50 mg/l to about 125 mg/l and,more preferably, from about 50 mg/l to about 100 mg/l.

One can find particular benefit in employing a support for themicrospores during culturing and subculturing. Any support thatmaintains the cells near the surface can be used. An illustrativeembodiment of a solid support is a TRANSWELL® culture dish. Anotherembodiment of a solid support for development of the microspores is abilayer plate wherein liquid media is on top of a solid base. Otherembodiments include a mesh or a millipore filter. Preferably, a solidsupport is a nylon mesh in the shape of a raft. A raft is defined as anapproximately circular support material which is capable of floatingslightly above the bottom of a tissue culture vessel, for example, apetri dish, of about a 60 or 100 mm size, although any other laboratorytissue culture vessel will suffice. In an illustrative embodiment, araft is about 55 mm in diameter.

Culturing isolated microspores on a solid support, for example, on a 10mm pore nylon raft floating on 2.2 ml of medium in a 60 mm petri dish,prevents microspores from sinking into the liquid medium and thusavoiding low oxygen tension. These types of cell supports enable theserial transfer of the nylon raft with its associatedmicrospore/embryoids ultimately to full strength medium containingactivated charcoal and solidified with, for example, GELRITE™(solidifying agent).

The liquid medium passes through the mesh while the microspores areretained and supported at the medium-air interface. The surface tensionof the liquid medium in the petri dish causes the raft to float. Theliquid is able to pass through the mesh; consequently, the microsporesstay on top. The mesh remains on top of the total volume of liquidmedium.

The culture vessels can be further defined as either (1) a bilayer 60 mmpetri plate wherein the bottom 2 ml of medium are solidified with 0.7%agarose overlaid with 1 mm of liquid containing the microspores; (2) anylon mesh raft wherein a wafer of nylon is floated on 1.2 ml of mediumand 1 ml of isolated microspores is pipetted on top; or (3) TRANSWELL®plates wherein isolated microspores are pipetted onto membrane insertswhich support the microspores at the surface of 2 ml of medium.

Examples of processes of tissue culturing and regeneration of corn aredescribed in, for example, European Patent Application 0 160 390, Greenand Rhodes (In: Maize for Biological Research, 367-372, 1982) and Duncanet al. (Planta, 165:322-332, 1985), Songstad et al. (Plant Cell Reports,7:262-265, 1988), Rao et al. (Maize Genetics Cooperation Newsletter, 60,1986), Conger et al. (Plant Cell Reports, 6:345-347, 1987), PCTApplication WO 95/06128, Armstrong and Green (Planta, 164:207-214,1985); Gordon-Kamm et al. (The Plant Cell, 2:603-618, 1990), and U.S.Pat. No. 5,736,369.

VII. Processes of Crossing Corn Plants and the Corn Plants Produced bySuch Crosses

The present invention provides processes of preparing novel corn plantsand corn plants produced by such processes. In accordance with such aprocess, a first parent corn plant may be crossed with a second parentcorn plant wherein the first and second corn plants are the parent linesof hybrid corn plant variety CH540328, or wherein at least one of theplants is of hybrid corn plant variety CH540328.

Corn plants (Zea mays L.) can be crossed by either natural or mechanicaltechniques. Natural pollination occurs in corn when wind blows pollenfrom the tassels to the silks that protrude from the tops of therecipient ears. Mechanical pollination can be effected either bycontrolling the types of pollen that can blow onto the silks or bypollinating by hand. In one embodiment, crossing comprises the steps of:

-   -   (a) planting in pollinating proximity seeds of a first and a        second parent corn plant, and preferably, seeds of a first        inbred corn plant and a second, distinct inbred corn plant;    -   (b) cultivating or growing the seeds of the first and second        parent corn plants into plants that bear flowers;    -   (c) emasculating flowers of either the first or second parent        corn plant, i.e., treating the flowers so as to prevent pollen        production, or alternatively, using as the female parent a male        sterile plant, thereby providing an emasculated parent corn        plant;    -   (d) allowing natural cross-pollination to occur between the        first and second parent corn plants;    -   (e) harvesting seeds produced on the emasculated parent corn        plant; and, where desired,    -   (f) growing the harvested seed into a corn plant, preferably, a        hybrid corn plant.

Parental plants are typically planted in pollinating proximity to eachother by planting the parental plants in alternating rows, in blocks orin any other convenient planting pattern. Where the parental plantsdiffer in timing of sexual maturity, it may be desired to plant theslower maturing plant first, thereby ensuring the availability of pollenfrom the male parent during the time at which silks on the female parentare receptive to pollen. Plants of both parental parents are cultivatedand allowed to grow until the time of flowering. Advantageously, duringthis growth stage, plants are in general treated with fertilizer and/orother agricultural chemicals as considered appropriate by the grower.

At the time of flowering, in the event that plant CH540328 is employedas the male parent, the tassels of the other parental plant are removedfrom all plants employed as the female parental plant to avoidself-pollination. The detasseling can be achieved manually but also canbe done by machine, if desired. Alternatively, when the female parentcorn plant comprises a cytoplasmic or nuclear gene conferring malesterility, detasseling may not be required. Additionally, a chemicalgametocide may be used to sterilize the male flowers of the femaleplant. In this case, the parent plants used as the male may either notbe treated with the chemical agent or may comprise a genetic factorwhich causes resistance to the emasculating effects of the chemicalagent. Gametocides affect processes or cells involved in thedevelopment, maturation or release of pollen. Plants treated with suchgametocides are rendered male sterile, but typically remain femalefertile. The use of chemical gametocides is described, for example, inU.S. Pat. No. 4,936,904, the disclosure of which is specificallyincorporated herein by reference in its entirety. Furthermore, the useof Roundup herbicide in combination with glyphosate tolerant corn plantsto produce male sterile corn plants is disclosed in PCT Publication WO98/44140.

Following emasculation, the plants are then typically allowed tocontinue to grow and natural cross-pollination occurs as a result of theaction of wind, which is normal in the pollination of grasses, includingcorn. As a result of the emasculation of the female parent plant, allthe pollen from the male parent plant is available for pollinationbecause tassels, and thereby pollen bearing flowering parts, have beenpreviously removed from all plants of the plant being used as the femalein the hybridization. Of course, during this hybridization procedure,the parental varieties are grown such that they are isolated from othercorn fields to minimize or prevent any accidental contamination ofpollen from foreign sources. These isolation techniques are well withinthe skill of those skilled in this art.

Both parental plants of corn may be allowed to continue to grow untilmaturity or the male rows may be destroyed after flowering is complete.Only the ears from the female parental plants are harvested to obtainseeds of a novel F₁ hybrid. The novel F₁ hybrid seed produced can thenbe planted in a subsequent growing season in commercial fields or,alternatively, advanced in breeding protocols for purposes of developingnovel inbred lines.

Alternatively, in another embodiment of the invention, one or both firstand second parent corn plants can be from variety CH540328. Thus, anycorn plant produced using corn plant CH540328 forms a part of theinvention. As used herein, crossing can mean selfing, backcrossing,crossing to another or the same variety, crossing to populations, andthe like. All corn plants produced using the corn variety CH540328 as aparent are, therefore, within the scope of this invention.

One use of the instant corn variety is in the production of hybrid seed.Any time the corn plant CH540328 is crossed with another, different,corn plant, a corn hybrid plant is produced. As such, hybrid corn plantcan be produced by crossing CH540328 with any second corn plant.Essentially any other corn plant can be used to produce a corn planthaving corn plant CH540328 as one parent. All that is required is thatthe second plant be fertile, which corn plants naturally are, and thatthe plant is not corn variety CH540328.

The goal of the process of producing an F₁ hybrid is to manipulate thegenetic complement of corn to generate new combinations of genes whichinteract to yield new or improved traits (phenotypic characteristics). Aprocess of producing an F₁ hybrid typically begins with the productionof one or more inbred plants. Those plants are produced by repeatedcrossing of ancestrally related corn plants to try to combine certaingenes within the inbred plants.

Corn has a diploid phase which means two conditions of a gene (twoalleles) occupy each locus (position on a chromosome). If the allelesare the same at a locus, there is said to be homozygosity. If they aredifferent, there is said to be heterozygosity. In a completely inbredplant, all loci are homozygous. Because many loci when homozygous aredeleterious to the plant, in particular leading to reduced vigor, lesskernels, weak and/or poor growth, production of inbred plants is anunpredictable and arduous process. Under some conditions, heterozygousadvantage at some loci effectively bars perpetuation of homozygosity.

A single cross hybrid corn variety is the cross of two inbred plants,each of which has a genotype which complements the genotype of theother. Typically, F₁ hybrids are more vigorous than their inbredparents. This hybrid vigor, or heterosis, is manifested in manypolygenic traits, including markedly improved yields, better stalks,better roots, better uniformity and better insect and diseaseresistance. In the development of hybrids only the F₁ hybrid plants aretypically sought. An F₁ single cross hybrid is produced when two inbredplants are crossed. A double cross hybrid is produced from four inbredplants crossed in pairs (A×B and C×D) and then the two F₁ hybrids arecrossed again (A×B)×(C×D).

Thousands of corn varieties are known to those of skill in the art, anyone of which could be crossed with corn plant CH540328 to produce ahybrid plant. For example, the U.S. Patent & Trademark Office has issuedmore than 300 utility patents for corn varieties. Estimates place thenumber of different corn accessions in gene banks around the world ataround 50,000 (Chang, In Plant Breeding in the 1990s, Stalker and Murphy(Eds.), Wallingford, U.K., CAB International, 17-35, 1992). The MaizeGenetics Cooperation Stock Center, which is supported by the U.S.Department of Agriculture, has a total collection of over 80,000individually pedigreed samples (http://maizecoop.cropsci.uiuc.edu/).

When the corn plant CH540328 is crossed with another plant to yieldprogeny, it can serve as either the maternal or paternal plant. For manycrosses, the outcome is the same regardless of the assigned sex of theparental plants. However, due to increased seed yield and productioncharacteristics, it may be desired to use one parental plant as thematernal plant. Some plants produce tighter ear husks leading to moreloss, for example due to rot. There can be delays in silk formationwhich deleteriously affect timing of the reproductive cycle for a pairof parental inbreds. Seed coat characteristics can be preferable in oneplant. Pollen can be shed better by one plant. Other variables can alsoaffect preferred sexual assignment of a particular cross.

The development of a hybrid corn variety involves three steps: (1) theselection of plants from various germplasm pools; (2) the selfing of theselected plants for several generations to produce a series of inbredplants, which, although different from each other, each breed true andare highly uniform; and (3) crossing the selected inbred plants withunrelated inbred plants to produce the hybrid progeny (F₁). During theinbreeding process in corn, the vigor of the plants decreases. Vigor isrestored when two unrelated inbred plants are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred plants is that the hybrid between any twoinbreds is always the same. Once the inbreds that give a superior hybridhave been identified, hybrid seed can be reproduced indefinitely as longas the homogeneity of the inbred parents is maintained. Conversely, muchof the hybrid vigor exhibited by F₁ hybrids is lost in the nextgeneration (F₂). Consequently, seed from hybrid varieties is not usedfor planting stock. It is not generally beneficial for farmers to saveseed of F₁ hybrids. Rather, farmers purchase F₁ hybrid seed for plantingevery year.

The development of inbred plants generally requires at least about 5 to7 generations of selfing. Inbred plants are then cross-bred in anattempt to develop improved F₁ hybrids. Hybrids are then screened andevaluated in small scale field trials. Typically, about 10 to 15phenotypic traits, selected for their potential commercial value, aremeasured. A selection index of the most commercially important traits isused to help evaluate hybrids. FACT, an acronym for Field AnalysisComparison Trial (strip trials), is an on-farm experimental testingprogram employed by Monsanto Company to perform the final evaluation ofthe commercial potential of a product.

During the next several years, a progressive elimination of hybridsoccurs based on more detailed evaluation of their phenotype. Eventually,strip trials (FACT) are conducted to formally compare the experimentalhybrids being developed with other hybrids, some of which werepreviously developed and generally are commercially successful. That is,comparisons of experimental hybrids are made to competitive hybrids todetermine if there was any advantage to further development of theexperimental hybrids. Examples of such comparisons are presentedhereinbelow. After FACT testing is complete, determinations may be madewhether commercial development should proceed for a given hybrid.

The present invention provides a genetic complement of the hybrid cornplant variety designated CH540328. As used herein, the phrase “geneticcomplement” means an aggregate of nucleotide sequences, the expressionof which defines the phenotype of a corn plant or a cell or tissue ofthat plant. By way of example, a corn plant is genotyped to determine arepresentative sample of the inherited markers it possesses. Markers arealleles at a single locus. They are preferably inherited in codominantfashion so that the presence of both alleles at a diploid locus isreadily detectable, and they are free of environmental variation, i.e.,their heritability is 1. This genotyping is preferably performed on atleast one generation of the descendant plant for which the numericalvalue of the quantitative trait or traits of interest are alsodetermined. The array of single locus genotypes is expressed as aprofile of marker alleles, two at each locus. The marker alleliccomposition of each locus can be either homozygous or heterozygous.Homozygosity is a condition where both alleles at a locus arecharacterized by the same nucleotide sequence or size of a repeatedsequence. Heterozygosity refers to different conditions of the gene at alocus. A preferred type of genetic marker for use with the invention issimple sequence repeats (SSRs), although potentially any other type ofgenetic marker could be used, for example, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),single nucleotide polymorphisms (SNPs), and isozymes.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

What is claimed is:
 1. A seed of the hybrid corn variety CH711103,produced by crossing a first plant of variety CV585594 with a secondplant of variety CV910316, wherein representative seed of said varietiesCV585594 and CV910316 have been deposited under ATCC Accession numbersPTA-10673 and PTA-11923, respectively.
 2. A plant of the hybrid cornvariety CH711103 grown from the seed of claim
 1. 3. A plant part of theplant of claim
 2. 4. The plant part of claim 3, further defined as anear, ovule, pollen or cell.
 5. A composition comprising the seed ofclaim 1 comprised in plant seed growth media.
 6. The composition ofclaim 5, wherein the growth media is soil or a synthetic cultivationmedium.
 7. A seed produced by crossing (a) a plant produced byintroducing a transgene into a plant of variety CV585594, or a selfedprogeny thereof, with a plant of variety CV910316, (b) a plant producedby introducing a transgene into a plant of variety CV910316, or a selfedprogeny thereof, with a plant of variety CV585594, or (c) a plantproduced by introducing a transgene into a plant of variety CV910316, ora selfed progeny thereof, with a plant produced by introducing atransgene into a plant of variety CV585594, or a selfed progeny thereof,wherein representative seed of said varieties CV585594 and CV910316 havebeen deposited under ATCC Accession numbers PTA-10673 and PTA-11923,respectively, and wherein the transgene is inherited by the seed.
 8. Theseed of claim 7, wherein the transgene confers a trait selected from thegroup consisting of male sterility, herbicide tolerance, insectresistance, disease resistance, waxy starch, modified fatty acidmetabolism, modified phytic acid metabolism, modified carbohydratemetabolism and modified protein metabolism.
 9. A method of producinghybrid corn seed, said method comprising crossing a plant of varietyCV585594 with a plant of variety CV910316, wherein representative seedof variety CV585594 and variety CV910316 have been deposited under ATCCAccession numbers PTA-10673 and PTA-11923, respectively.
 10. A seed ofthe hybrid corn variety CH711103 comprising at least a first singlelocus conversion, produced by crossing a first plant of variety CV585594with a second plant of variety CV910316, wherein representative seed ofsaid varieties CV585594 and CV910316 have been deposited under ATCCAccession numbers PTA-10673 and PTA-11923, respectively, and wherein oneor both of the first plant and second plant comprises the single locusconversion.
 11. The seed of claim 10, wherein the single locusconversion confers a trait selected from the group consisting of malesterility, herbicide tolerance, insect resistance, disease resistance,waxy starch, modified fatty acid metabolism, modified phytic acidmetabolism, modified carbohydrate metabolism and modified proteinmetabolism.
 12. A plant grown from the seed of claim
 10. 13. A method ofintroducing a heritable trait into hybrid corn variety CH711103, saidmethod comprising the steps of: (a) introducing at least a firstheritable trait into at least one inbred corn variety selected from thegroup consisting of variety CV585594 and variety CV910316 to produce aplant of the first inbred corn variety that heritably carries the trait,wherein the heritable trait is introduced into said first inbred cornvariety by backcrossing and wherein representative samples of seed ofvariety CV585594 and variety CV910316 have been deposited under ATCCAccession numbers PTA-10673 and PTA-11923, respectively; and (b)crossing a plant of the first inbred corn variety that heritably carriesthe trait with a plant of a different variety selected from said groupconsisting of CV585594 and CV910316 to produce a plant of hybrid cornvariety CH711103 comprising the heritable trait.
 14. The method of claim13, wherein the trait is selected from the group consisting of malesterility, herbicide tolerance, insect resistance, disease resistance,waxy starch, modified fatty acid metabolism, modified phytic acidmetabolism, modified carbohydrate metabolism and modified proteinmetabolism.
 15. The method of claim 14, further comprising repeatingstep (a) at least once to introduce at least a second heritable traitinto hybrid corn variety CH711103, wherein the second heritable trait isselected from the group consisting of male sterility, herbicidetolerance, insect resistance, disease resistance, waxy starch, modifiedfatty acid metabolism, modified phytic acid metabolism, modifiedcarbohydrate metabolism and modified protein metabolism.
 16. A plantproduced by the method of claim
 13. 17. A method of producing a cornplant derived from the hybrid corn variety CH711103, wherein the methodcomprises crossing the plant of claim 2 with a second corn plant toproduce a progeny corn plant derived from the hybrid corn varietyCH711103.
 18. The method of claim 17, further comprising the steps of:(a) crossing the progeny corn plant derived from the hybrid corn varietyCH711103 with itself or a second plant to produce a seed of a progenyplant of a subsequent generation; (b) growing a progeny plant of asubsequent generation from the seed and crossing the progeny plant of asubsequent generation with itself or a second plant; and (c) repeatingsteps (a) and (b) for at least an additional 3-10 generations to producea corn plant further derived from the hybrid corn variety CH711103. 19.The method of claim 18, further comprising the step of: (d) crossing thecorn plant further derived from the hybrid corn variety CH711103 with asecond, distinct corn plant.
 20. A method of producing a commodity plantproduct, said method comprising obtaining the plant of claim 2 or a partthereof and producing said commodity plant product therefrom.
 21. Themethod of claim 20, wherein the commodity plant product is grain,starch, seed oil, corn syrup or protein.